The Flavivirus genus incorporates over 60 closely related viruses including several human pathogens of global and local epidemiological importance, with most of them being transmitted by arthropod vectors. West Nile (WN) virus is the most widespread of the flaviviruses, with geographic distribution now including four out of five continents. Given its adaptability and diversified transmission cycles, this virus, having been appeared on the American continent, very likely will integrate into the existing ecosystems resulting in establishment of a permanent and substantial epidemiological concern at the national level. The broad geographic distribution of West Nile virus has resulted in considerable heterogeneity among strains forming two distinct WN virus lineages, pathogenic properties of which remain largely uncharacterized. While lineage I virus strains were often identified in connection with human or equine outbreaks, strains of the second lineage more often were identified by occasion as field isolates. The WN virus emerged in the U.S. also belongs to lineage I and has been associated with neurological disease both in birds and humans. This strain also appeared highly cytopathic in cell culture. We have identified several highly cytopathic and non-cytopathic WN strains, which belong to lineage I and II, respectively. We intend to compare cyto- and pathogenic properties of these strains in cell culture and in the mouse model. Using a 385-99 isolate as a representative, we will assemble an infectious clone of lineage I West Nile virus. Using this infectious clone and the lineage II WN infectious clone that we have reported earlier, we will prepare chimeric viruses to determine if the envelope protein genes carry determinants responsible for the observed pathogenicity in mice and cytopathology in cell culture and in vivo. Data obtained in the study will contribute to characterization of the infectious agent recently emerged in the U.S. and will aid in rational design of anti-WN vaccines and antivirals.